1. Field
The invention is in the field of staining reagents for staining hematology samples using automated staining equipment and in methods used for such staining, particularly in the field generally referred to as Romanowsky staining, which includes variations such as Wright and Giemsa staining.
2. State of the Art
Automated slide staining equipment is currently commercially available. One such stainer is manufactured by Wescor, Inc. of Logan, Utah, and is sold under the name Areospray®. In such stainers, slides to be stained are placed in a slide carousel which is rotated in a spray chamber by a motor. One or more spray nozzles are positioned in the spray chamber adjacent the carousel. During rotation of the carousel, the slides are sprayed with various reagents as they move past the spray nozzles. Pumps are provided to pump reagents from reagent reservoirs to the nozzles which atomize the reagents and spray them onto the slides as the slides pass the nozzles. Such staining equipment is shown, for example, in U.S. Pat. Nos. 5,009,185 and 5,180,606. Automatic staining equipment can be used to perform many types of staining activities, i.e., to carry out various embodiments of various different staining methods, and various different staining reagents are used in performing the many types of staining activities.
Each particular type of staining activity or staining method uses particular reagents such as particular staining and rinsing reagents, and the equipment goes through particular steps to apply the particular reagents in particular orders and amounts while the carousel rotates at particular speeds. The equipment is programmed, either at the factory or by the user, to carry out a particular staining method. The program directs the equipment to draw reagents from particular reagent reservoirs (the user has to place the correct reagents in the correct reservoirs) at particular times and controls both the spraying time and carousel speed to perform the particular method programmed. Usually the equipment is programmed so the user can select between several variations of the method and may be able to select or set one or more of the variables.
In the field of hematology sample staining, there is a type of staining referred to generally as Romanowsky staining. Romanowsky is generally credited with discovery of the unique effects of the thiazin-eosin blood stain in 1891, D. Romanowsky (1891) Zur Frage der Parisitologie und Therapie der Malaria. St Petersb. med. Wschr. 16, 297-302. and D. Romanowsky (1891) Zur Frage der Parisitologie und Therapie der Malaria. St Petersb. med. Wschr 16, 307-315. Since that time many variants of Romanowsky staining have been proposed, P. N. Marshall (1978) Romanowsky-type stains in hematology. Histochem. J. 10, 1-29. Popular modem methods are those developed by Wright in the U.S. and Giemsa in Europe. Combination stains such as Wright Giemsa and May-Grünwald Giemsa (Pappenheim) are also widely used. The staining stock solutions for these methods are typically thiazin-eosinates dissolved in organic solvents, principally methanol for Wright and methanol/glycerol for Giemsa. These stains contain no water, but must be diluted with water for final staining to occur. Staining with an alcohol concentration of greater than about 30% slows staining dramatically and completely inhibits the formation of the purple coloration of nuclei (the Romanowsky-Giemsa effect (RGE)). The working stains prepared by diluting the stock solutions are unstable and must be prepared shortly before use. Yip et al., U.S. Pat. No. 4,595,524, avoided stability problems by using separated dye solutions, but mixed them prior to staining as did Romanowsky in the original development of blood staining. The blended Yip stain is primarily aqueous with small amounts of acetone (13%) and dimethyl formamide (6.5%) to reduce precipitation. It is essentially equivalent to a Wright Giemsa stain which has been diluted with buffer for the final staining step.
A rapid methodology is employed in the Wescor Aerospray® 7120 Hematology Stainer which sequentially stains with separate aqueous solutions. Several such stains are available from commercial suppliers. The first stain contains eosin; the second contains thiazin dyes. Keeping the dyes separated avoids stain precipitation and the aqueous solvent allows very rapid staining.
During the course of developing a Romanowsky type staining procedure for the Aerospray® 7120 Hematology Stainer, the absence of basophil granules in the resulting smears was recognized, along with other differences in staining results when compared with the traditional stains. The granules of basophilic leukocytes (basophils) primarily contain histamine and heparin which are small molecules that are highly water soluble. In Wright or Giemsa staining these granules are stabilized by simultaneous binding of thiazin (primarily azure B) and eosin molecules from an aqueous organic solvent suitable for granule preservation. When well stabilized they produce a distinctive appearance. The cell is filled with large blue black granules making identification easy and certain. When the granules are absent, they are easily confused with other cell types unless the observer receives specialized instructions.
In order to stabilize the basophil granules in the Aerospray® 7120, Wescor developed a predip fixative (Basofix®) containing azure B, polyvinylpyrrolidone, methanol, and less than 4% water. Slides are dipped into the solution and then allowed to dry, thereby depositing a film of azure B and PVP on the slide. The basophil granules appear to stain when the eosin stain is applied by the Aerospray® 7120, presumably by producing thiazin-eosinate at the slide surface. Efforts to fix with Basofix inside the instrument produced rather poor results, granules were only half as good as for slides manually fixed outside the instrument. In addition, other cellular granulation such as the primary granulation of immature leukocytes and granulation in segmented neutrophils were not adequately improved whether the fixation was automated or manual. This led to the conclusion that optimal automated staining would require a mixed thiazin-eosin stain for stabilizing the basophils and other granules.
Considerable difficulty was encountered in adapting a Wright/Giemsa application to the Aerospray® 7120. A commercial rapid Wright stain was run on the Aerospray® 7120. In hand staining, this stain is applied to the blood smear in anhydrous form and then the smear is transferred to water to complete the staining reaction. This accomplishes the necessary aqueous dilution. While staining was close to usable in the Aerospray® 7120, after program optimization of alternate stain and water sprays, the basophils were totally degranulated. This was very surprising since the thiazin-eosinate dye combination was expected to stabilize and stain the granules.
FIG. 1 shows that the presence of water is necessary to fix the basophil granules. Slides were placed in a commercial Wright stain, such as made by Harleco, containing varying amounts of water for one minute, then centrifuged in the Aerospray® 7120 for 30 seconds to remove excess stain and dry the slides. The extent of granule preservation was estimated under a 100× oil immersion lense using a light microscope. In the absence of water very little preservation is evident, but granulation increases markedly as the water content is increased.
During hand staining procedures with Wright stain, slides are typically fixed in the anhydrous stain to cause the cells to adhere to the glass slide and stabilize their morphology. This must be done with methanol or anhydrous staining reagent since the presence of water causes cell destruction, cloudiness and water artifacts in the smear. Once fixed, the slides are transferred to a diluted (5 to 100 fold) stain to allow final staining to occur. In such environments, basophil granules dissolve rapidly if not previously stabilized. The above information suggests that in hand staining, the transition from anhydrous stain to aqueous is gradual enough to produce conditions favorable for basophil stabilization. When slides are transferred from the anhydrous stain to a dilute one, the film of stain which is immobilized by the slide is apparently diluted slowly enough to allow the fixation of the basophil granules. In the Aerospray®, the force of the spray droplets impacting the slide appears to penetrate the thin layer of stain held by the vertical slide. This penetration mixes and quickly washes away the dyes thus preventing adequate staining and stabilization of the basophil granules during the rapid transition from anhydrous to diluted stain. During efforts to optimize basophil fixation and staining, a good deal of unpredictable behavior was observed in basophil granulation results. While basophil granules were stabilized, results were nearly always inferior to the simple manual procedure described above. The need remains for a staining procedure or method usable with automatic staining equipment to provide good basophil granulation and good Romanowsky type staining.